Vol 9. Issue 8 / March 9, 2009
The Conqueror Worm
By Eric Sauter
Scientists from The Scripps Research Institute and other institutions have found that a deep sea worm, Alvinella pompejana, may offer highly stable human-like proteins that could ultimately provide a far more effective model for study than proteins from their simpler and nuclei-free relatives now in use. This discovery could prove to be uniquely valuable in increasing our understanding of amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, a progressive neurodegenerative disease that ravages neurons in the brain and spinal cord.
The new study—led by Professor John Tainer of Scripps Research and its Skaggs Institute for Chemical Biology and S. Craig Cary of the University of Delaware and the University of Waikato, New Zealand—was published in the February 6, 2009 (Volume 385, Issue 5) of the Journal of Molecular Biology.
A Strange Creature
The deep sea worm Alvinella pompejana, discovered by French researchers in the early 1980s—lives several miles below the surface and is considered the hottest animal on the planet. The creature lives in hydrothermal vents, known as "black smoker chimneys," off the Pacific coast of Costa Rica in temperatures averaging as high as 68 °C, with spikes up to 84 °C—that is, 176° F.
The creatures are charmingly ugly, with what appear to be gray hairs covering nearly all of their quarter-foot-long bodies and rust colored tentacle-like gills on their heads. Those grey hairs are actually bacteria that feed on mucus extruded from glands beneath the worm's skin. The bacteria may also help the worms survive the extreme temperatures, providing a kind of thermal blanket that protects them or at least their tails, which the animals keep buried in the hottest part of the vents while their heads bask in the cooler waters outside.
One of the characteristics that makes these worms so interesting to scientists is that they are eukaryotes—their cells have nuclei, as opposed to prokaryotes such as bacteria whose cells do not—making comparisons to human proteins more direct.
"While most of the microbes have similar proteins, eukaryotes lack important regulatory mechanisms," said Tainer. "This organism is closer to us than to bacteria and can bridge the gap."
The story of collecting the worms for the research is another tale in itself (see "Way Down Below the Ocean" in this week's News&Views).
Once the samples were in the lab, the scientists developed a copy of the worm's DNA for sequencing, cloning, and protein expression, and solved the crystal structure of the worm's superoxide dismutase (SOD) enzyme. The scientists found that the A. pompejana SOD is not only strikingly similar to the human form of the protein, it is remarkably stable, even more stable than its human equivalent.
"Human SOD proteins are highly stable in the first place, so to find one that is actually more stable than that is even more impressive," said David Shin, a senior research associate in the Tainer laboratory and first author of the study. "What is highly beneficial is that both the human and worm proteins are similar in overall structure and in primary amino acid sequence making subtle stabilizing traits more readily identifiable."
The enzyme superoxide dismutase (SOD) is a natural antioxidant—converting or changing the superoxide radicals (oxygen with an added electron) into oxygen and hydrogen peroxide, which makes SOD an important antioxidant defense system in nearly all cells exposed to oxygen radicals.
SOD has applications in cancer, which produces substantially more oxidants than regular cells. Inhibiting SOD damages the cancer cells—they basically overdose on oxidants—and controls their growth. As a result, SOD is an important target protein for drug development.
In addition, according to Tainer, SOD is mutated in some 20 percent of cases of inherited or familial cases of ALS.
The stability of A. pompejana SOD, the study noted, may be the result of a reduction in certain molecular movements in the worm protein, movements that increase the destructive interactions associated with ALS.
"With ALS, in a substantial number of cases, when you destabilize the protein amyloid-like fiber aggregations are formed," said Shin. "By using the worm protein we might be able to tease out what the mechanisms are behind some of these mutations that initiate this. It's a very good start towards deciphering the process."
Deciphering the Process
In the new study, comparative structural analyses of eukaryotic SODs identified amino acid residues that may contribute to the enhanced stability of the A. pompejana SOD over human SOD. The scientists identified several interactions that anchored the structural elements of the protein, promoting stability by maintaining the protein's natural fold and assembly (proteins perform their normal functions in a specifically folded state).
"We were able to examine catalysis and various features that promote stability," Tainer said. "The hydrogen peroxide co-crystal structure—which is a first—is very important for understanding the enzymatic mechanism. All the previously proposed mechanisms were based on structures that did not contain superoxide or hydrogen peroxide. Our structure clarifies the details, allows us for the first time to actually see the points of contact."
More generally, the study's findings suggest that this functional protein stability can be substantially improved (or degraded) by substitutions or mutations that increase (or decrease) the rigidity of the weakest parts of the structure, without altering the protein's core fold or assembly. These weak links may actually induce protein unfolding and structural destabilization that could trigger the aggregation of amyloid-like filaments in ALS and the onset of the disease.
As a result of the new work, there is now an important test model to study destabilizing mutations that occur in ALS and how they play out. In addition, this work spurred the search for other stable worm proteins that are representative of human proteins that play key roles in disease. So a large-scale Alvinella DNA sequencing project is underway via a collaboration between the group and the generous efforts of the Joint Genome Institute's Community Sequencing Program.
In addition to Tainer, Cary, and Shin, authors of the paper, "Superoxide Dismutase Structures: Stability, Mechanism, and Insights into the Human Disease Amyotrophic Lateral Sclerosis from Eukaryotic Thermophile Alvinella pompejana," are Michael DiDonato, David P. Barondeau, Chiharu Hitomi and Elizabeth D. Getzoff of The Scripps Research Institute; Greg L. Hura of Lawrence Berkeley National Laboratory; and J. Andrew Berglund of the University of Oregon. See http://dx.doi.org/10.1016/j.jmb.2008.11.031.
The study was supported by the 3rd Annual Incyte Discovery Award, the National Institutes of Health, the National Sciences Foundation, the Department of Energy, and The Skaggs Institute for Chemical Biology, and Ruth L. Kirschstein National Research Service Awards Fellowships.
Send comments to: mikaono[at]scripps.edu